Method for Isolation of Nucleic Acid Containing Particles and Extraction of Nucleic Acids Therefrom

ABSTRACT

A method for extracting nucleic acids from a biological sample by isolating nucleic acid-containing particles from the biological sample by one or more centrifugation procedures, performing one or more steps to mitigate adverse factors that prevent or might prevent high quality nucleic acid extraction, and extracting nucleic acids from the isolated particles. The centrifugation procedures are performed at a speed not exceeding about 200,000 g. The extracted nucleic acids contain both 18S and 28S rRNA.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/883,673, which is a national stage application, filed under 35 USC§371, of PCT Application No. PCT/US2011/060251, filed Nov. 10, 2011,which claims priority to U.S. provisional application No. 61/412,369,filed Nov. 20, 2010, the contents of each of which are incorporatedherein by reference in their entireties.

FIELD OF INVENTION

The present invention relates to the general fields of nucleic acidextraction from a biological sample, particularly the isolation ofnucleic acid-containing particles from body fluids and extraction ofnucleic acids from the isolated particles.

BACKGROUND

Small microvesicles shed by cells are often described as “exosomes”(Thery et al., 2002). Exosomes are reported as having a diameter ofapproximately 30-100 nm and are shed from many different cell typesunder both normal and pathological conditions (Thery et al., 2002).Exosomes are classically formed from the inward invagination andpinching off of the late endosomal membrane. This results in theformation of a multivesicular body (MVB) laden with small lipid bilayervesicles, each of which contains a sample of the parent cell's cytoplasm(Stoorvogel et al., 2002). Fusion of the MVB with the cell membraneresults in the release of these exosomes from the cell, and theirdelivery into the blood, urine, cerebrospinal fluid, or other bodilyfluids.

Another category of cell-derived microvesicles are formed by directlybudding off of the cell's plasma membrane, are usually larger in sizethan exosomes, and like exosomes, also contain a sample of the parentcell's cytoplasm (Cocucci et al., 2009) (Orozco and Lewis, 2010).

Recent studies reveal that nucleic acids within microvesicles have arole as biomarkers. For example, WO 2009/100029 describes, among otherthings, the use of nucleic acids extracted from microvesicles in GBMpatient serum for medical diagnosis, prognosis and therapy evaluation.WO 2009/100029 also describes the use of nucleic acids extracted frommicrovesicles in human urine for the same purposes. The use of nucleicacids extracted from microvesicles is considered to potentiallycircumvent the need for biopsies, highlighting the enormous diagnosticpotential of microvesicle biology (Skog et al., 2008).

Several methods of isolating microvesicles from a biological sample havebeen described in the art. For example, a method of differentialcentrifugation is described in a paper by Raposo et al. (Raposo et al.,1996), a paper by Skog et. al. (Skog et al., 2008) and a paper byNilsson et. al. (Nilsson et al., 2009). Methods of anion exchange and/orgel permeation chromatography are described in U.S. Pat. Nos. 6,899,863and 6,812,023. Methods of sucrose density gradients or organelleelectrophoresis are described in U.S. Pat. No. 7,198,923. A method ofmagnetic activated cell sorting (MACS) is described in a paper by Taylorand Gercel-Taylor (Taylor and Gercel-Taylor, 2008). A method ofnanomembrane ultrafiltration concentration is described in a paper byCheruvanky et al. (Cheruvanky et al., 2007). A method of Percollgradient isolation is described in a publication by Miranda et. al(Miranda et al., 2010). Further, microvesicles may be identified andisolated from bodily fluid of a subject by a microfluidic device (Chenet al., 2010).

In research and development, as well as commercial applications ofnucleic acid biomarkers, it is desirable to extract high quality nucleicacids from biological samples in a consistent, reliable, and practicalmanner. An object of the present invention is therefore to provide amethod for quick and easy isolation of nucleic acid-containing particlesfrom biological samples such as body fluids and extraction of highquality nucleic acids from the isolated particles. The method of theinvention may be suitable for adaptation and incorporation into acompact device or instrument for use in a laboratory or clinicalsetting, or in the field.

SUMMARY

The present invention is based on our discovery that low speedcentrifugation can be used to pellet particles from a biological sampleand extract high quality nucleic acids from the particles. In oneaspect, the invention is a method for extracting nucleic acids byisolating nucleic acid-containing particles from a biological sample byone or more centrifugation procedures at a speed not exceeding about200,000 g, performing one or more steps to mitigate adverse factors thatprevent or might prevent high quality nucleic acid extraction; andextracting nucleic acids from the isolated particles.

In some embodiments, the centrifugation procedures are performed atspeeds of about 2,000 g to about 200,000 g. In other embodiments, thecentrifugation procedures are performed at speeds not exceeding about50,000 g. In still other embodiments, the centrifugation procedures areperformed at speeds not exceeding about 20,000 g. In some embodiments,the method is used to extract nucleic acids from microvesicles,RNA-protein complexes, DNA-protein complexes, or a combination of any ofmicrovesicles, RNA-protein complexes, and DNA-protein complexes.

In some embodiments, the biological sample is a body fluid, for example,a serum or a urine sample from a subject. The subject, for example, canbe a human or other mammal. The extracted nucleic acids can be RNA, DNA,or both RNA and DNA. In some further embodiments, the nucleic acids thusextracted contain one or more polynucleotides which are more than 90%homologous to a nucleic acid sequence corresponding to EGFR, BRAF, KLK3,18S, GAPDH, HPRT1, GUSB, ACTB, B2M, RPLP0, HMBS, TBP, PGK1, IJBC, PPIA,ALCAM, C5AR1, CD160, CD163, CD19, CD1A, CD1C, CD1D, CD2, CD209, CD22,CD24, CD244, CD247, CD28, CD37, CD38, CD3D, CD3G, CD4, CD40, CD40LG,CD5, CD6, CD63, CD69, CD7, CD70, CD72, CD74, CD79A, CD79B, CD80, CD83,CD86, CD8A, CD8B, CD96, CHST10, COL1A1, COL1A2, CR2, CSFIR, CTLA4, DPP4,ENG, FAS, FCER1A, FCER2, FCGR1A/FCGR1B/FCGR1C, HLA-A/HLA-A29.1, HLA-DRA,ICAM2, IL12RB1, IL1R2, IL2RA, ITGA1, ITGA2, ITGA3, KLRB1, KLRC1, KLRD1,KRT18, KRT5, KRT8/LOC728638, MS4A1, MYH10, MYH9, MYOCD, NCAM1, NOS3,NT5E, PECAM1, RETN, S100A8, SELP, ST6GAL1, EPCAM, TEK, TNFRSF4, TNFRSF8,TPSAB1/TPSB2, VCAM1, or VWF.

In some embodiments, 18S rRNA and 28S rRNA are detectable in theextracted nucleic acids. In some instances, the ratio of the amount of18S rRNA to the amount of 28S rRNA as detected in the extracted nucleicacids is about 0.5 to about 1.0. In other instances, the ratio of theamount of 18S rRNA to the amount of 28S rRNA as detected in theextracted nucleic acids is about 0.5.

In some embodiments, the step of performing one or more steps tomitigate adverse factors is achieved by treating the biological sampleand/or the isolated particles with DNase, RNase inhibitor, or both DNaseand RNase inhibitors. In certain embodiments, the step of performing oneor more steps to mitigate adverse factors is achieved by a step oftreating the biological sample with RNase inhibitor before isolating theparticles.

In another aspect, the present invention is a nucleic acid sampleobtained from a biological sample by the any of above described methods.The nucleic acid sample thus obtained can be used in variousapplications. In some embodiments, the above method and resultingnucleic acid sample are used for aiding in the diagnosis of a subject bydetermining the presence or absence of a biomarker within the nucleicacid sample that is associated with a known disease or other medicalcondition. In other embodiments, the above method and resulting nucleicacid sample are used for monitoring the progress or reoccurrence of adisease or other medical condition in a subject by determining thepresence or absence of a biomarker with in the sample that is associatedwith the progress or reoccurrence of a known stage or the reoccurrenceof a disease or other medical condition. In still other embodiments, theabove method and resulting nucleic acid sample are used in theevaluation of treatment efficacy for a subject undergoing orcontemplating treatment for a disease or other medical condition bydetermining the presence or absence ofa biomarker within the sample thatis associated with treatment efficacy for the subject undergoing orcontemplating treatment for a disease or other medical condition.

In some further embodiments, the biomarker detected in the aboveapplications is a nucleic acid corresponding to any one or more of thegenes consisting of EGFR, BRAF, KLK3, 18S, GAPDH, HPRT1, GUSB, ACTB,B2M, RPLP0, HMBS, TBP, PGK1, UBC, PPIA, ALCAM, C5AR1, CD160, CD163,CD19, CD1A, CDIC, CD1D, CD2, CD209, CD22, CD24, CD244, CD247, CD28,CD37, CD38, CD3D, CD3G, CD4, CD40, CD40LG, CD5, CD6, CD63, CD69, CD7,CD70, CD72, CD74, CD79A, CD79B, CD80, CD83, CD86, CD8A, CDSB, CD96,CHST10, COL1A1, COL1A2, CR2, CSF1R, CTLA4, DPP4, ENG, FAS, FCER1A,FCER2, FCGR1A/FCGR1B/FCGR1C, HLA-A/HLA-A29.1, HLA-DRA, ICAM2, IL12RB1,IL1R2, IL2RA, ITGA1, ITGA2, ITGA3, KLRB1, KLRC1, KLRD1, KRT18, KRT5,KRT8/LOC728638, MS4A1, MYH10, MYH9, MYOCD, NCAM1, NOS3, NT5E, PECAM1,RETN, S100A8, SELP, ST6GAL1, EPCAM, TEK, TNFRSF4, TNFRSF8, TPSAB1/TPSB2,VCAM1, and VWF.

Yet another aspect of the invention is a kit for use in the abovemethods. The kit may include RNase inhibitor in a quantity sufficient tomitigate adverse factors that prevent or might prevent high qualitynucleic acid extraction, and an RNA purification reagent. The kit mayoptionally further include a lysis buffer, DNase, or instructions forusing the kit and reagent in it in the extraction of nucleic acids fromisolated particles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C are Bioanalyzer plots depicting the analysis ofnucleic acids extracted from particles isolated from serum samples asdescribed in Examples 1, 2 and 3, respectively. The pseudogel in FIG. 1Adepicts the content of the same nucleic acid extraction as depicted inthe Bioanalyzer plot of FIG. 1A. The plots and the pseudogel weregenerated by an RNA pico chip run on an Agilent Bioanalyzer.

FIG. 2 is a Bioanalyzer plot depicting the analysis of nucleic acidsextracted from particles isolated from a urine sample, as described inExample 5 below, and a pseudogel depicting the content of the samenucleic acid extraction. The plot and the pseudogel were generated by anAgilent Bioanalyzer.

FIG. 3 is a Bioanalyzer plot depicting the analysis of nucleic acidsextracted from particles isolated from serum samples in group A, Example4 (20,000 g centrifugation speed).

FIG. 4 is a Bioanalyzer plot depicting the analysis of nucleic acidsextracted from particles isolated from serum samples in group B, Example5 (120,000 g centrifugation speed).

FIG. 5 is a plot depicting the comparison of Ct values for genesanalyzed with the Taqman PCR array as between group A (Y-axis) and groupB (X-axis) in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

As described above, cell-derived vesicles are heterogeneous in size withdiameters ranging from about 10 nm to about 5000 nm. For example,“exosomes” have diameters of approximately 30 to 100 nm, with sheddingmicrovesicles and apoptotic bodies often described as larger (Orozco andLewis, 2010). Exosomes, shedding microvesicles, microparticles,nanovesicles, apoptotic bodies, nanoparticles and membrane vesiclesco-isolate using various techniques and will, therefore, collectively bereferred to throughout this specification as “microvesicles” unlessotherwise expressly denoted.

Other nucleic acid-containing particles, e.g., RNA-protein complexes andDNA-protein complexes, may co-isolate with microvesicles using thevarious methods and techniques described herein. Accordingly, thegeneric term “particles” will be used herein to refer to microvescles,RNA-protein complexes, DNA-protein complexes, and any other nucleicacid-containing particles that could be isolated according to themethods and techniques described herein. The methods and techniquesdescribed herein are equally applicable to the isolation of RNA-proteincomplexes, DNA-protein complexes, or other nucleic acid-containingparticles, and microvesicles of all sizes (either as a whole, as selectsubsets, or as individual species).

The present invention is partly based on the discovery that lowercentrifugation speeds can achieve similar results as highercentrifugation speeds during nucleic acid-containing particle isolation.As such, in one aspect, the present invention is directed to novelmethods for isolating particles from a biological sample and extractingnucleic acids from the isolated particles. The nucleic acid extractionsobtained by the methods described herein may be useful for variousapplications in which high quality nucleic acid extractions are requiredor preferred.

As used herein, the term “high quality” in reference to nucleic acidextraction means an extraction in which one is able to detect 18S and28S rRNA, preferably in a ratio of approximately 1:1 to approximately1:2; and more preferably, approximately 1:2. Ideally, high qualitynucleic acid extractions obtained by the methods described herein willalso have an RNA integrity number of greater than or equal to 5 for alow protein biological sample (e.g., urine), or greater than or equal to3 for a high protein biological sample (e.g., serum), and a nucleic acidyield of greater than or equal to 50 pg/ml from a 20 ml low proteinbiological sample or a 1 ml high protein biological sample.

High quality RNA extractions are desirable because RNA degradation canadversely affect downstream assessment of the extracted RNA, such as ingene expression and mRNA analysis, as well as in analysis of non-codingRNA such as small RNA and microRNA. The new methods described hereinenable one to extract high quality nucleic acids from particles isolatedfrom a biological sample so that an accurate analysis of nucleic acidswithin the particles can be carried out.

Broadly described, the novel methods include, for example, the steps ofobtaining a biological sample; isolating nucleic acid-containingparticles from the biological sample by one or more centrifugationsteps; mitigating or removing adverse factors that prevent high qualityextraction of nucleic acids from the sample; and extracting nucleicacids from the isolated particles; followed, optionally, by nucleic acidanalysis. The centrifugation step or steps may be performed atrelatively low speeds as compared to traditional methods of isolatingparticles from biological samples by centrifugation. None of thecentrifugation steps in the inventive methods described herein mayexceed about 200,000 g.

Suitable centrifugation speeds are up to about 200,000 g; for examplefrom about 2,000 g to less than about 200,000 g. Speeds of above about15,000 g and less than about 200,000 g or above about 15,000 g and lessthan about 100,000 g or above about 15,000 g and less than about 50,000g are preferred. Speeds of from about 18,000 g to about 40,000 g orabout 30,000 g; and from about 18,000 g to about 25,000 g are morepreferred. Particularly preferred is a centrifugation speed of about20,000 g.

The methods described herein may be used with a variety of commerciallyavailable centrifuge machines and for the purpose of isolating variousspecies of particles. A person of skill in the art will be able to usethe well known K-factor to optimize the centrifugation parameters for aparticular centrifuge device selected for use in the method. Forexample, the K-factor, which denotes the clearing factor of a centrifugerotor at maximum rotation speed, may be used to determine the time (“T”)required for pelleting a fraction with a known sedimentation coefficient(“S”). The lower the K-factor, the more efficient the pelleting with anygiven centrifuge device. The K-factor can be calculated by the followingformula:

K=2.53*10¹¹*ln(r _(max) /r _(min))]/RPM²,

wherein r_(max) is the maximum radius from the centrifuge's axis ofrotation, and r_(min) is the minimum radius from the axis of rotation.The r_(max) and r_(min) are usually available from the centrifugemanufacturer. RPM is the speed in revolutions per minute. The K-factoris related to the sedimentation coefficient S by the formula:

T=K/S,

where T is the time to pellet a certain particle in hours. Where S is aknown constant for a certain particle, this relationship can be used tointerconvert between different rotors using the following formula:

T ₁ /K ₁ =T ₂ /K ₂,

where T₁ is the time to pellet in one rotor, and K₁ is the K-factor ofthat rotor, K₂ is the K-factor of the other rotor, and T₂, the time topellet in the other rotor. If one knows K₁, T₁, and can calculate K₂,then T₂ may be determined. In this manner, one does not need access tothe exact centrifuge rotor cited in a particular protocol, as long asthe K-factor can be calculated. If the sedimentation constant (S) isunknown for a particular substance to be pelleted, then one of skill inthe art may determine T₂ based on empirical data as to T₁ for the samesubstance and calculation of K₂ for the different rotor.

Generally, suitable K factors are within the range of about 300 to about1000; preferably within the range of about 400 to about 600; and morepreferably about 520.

Generally, suitable times for centrifugation are from about 5 minutes toabout 2 hours, for example, from about 10 minutes to about 1.5 hours, ormore preferably from about 15 minutes to about 1 hour. A time of about0.5 hours is sometimes preferred.

It is sometimes preferred to subject the biological sample tocentrifugation at about 20,000 g for about 0.5 hours. However the abovespeeds and times can suitably be used in any combination (e.g., fromabout 18,000 g to about 25,000 g, or from about 30,000 g to about 40,000g for about 10 minutes to about 1.5 hours, or for about 15 minutes toabout 1 hour, or for about 0.5 hours, and so on).

The centrifugation step or steps may be carried out at below-ambienttemperatures, for example at about 0-10° C., preferably about 1-5° C.,e.g., about 3° C. or about 4° C.

As used herein, the term “biological sample” refers to a sample thatcontains biological materials such as a DNA, a RNA and a protein. Insome embodiments, the biological sample may suitably comprise a bodilyfluid from a subject. The bodily fluids can be fluids isolated fromanywhere in the body of the subject, preferably a peripheral location,including but not limited to, for example, blood, plasma, serum, urine,sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nippleaspirates, lymph fluid, fluid of the respiratory, intestinal, andgenitourinary tracts, tear fluid, saliva, breast milk, fluid from thelymphatic system, semen, cerebrospinal fluid, intra-organ system fluid,ascitic fluid, tumor cyst fluid, amniotic fluid and combinationsthereof. In some embodiments, the preferred body fluid for use as thebiological sample is urine. In other embodiments, the preferred bodyfluid is serum. In still other embodiments, the preferred body fluid iscerebrospinal fluid.

Suitably a sample volume of about 0.1 ml to about 30 ml fluid may beused. The volume of fluid may depend on a few factors, e.g., the type offluid used. For example, the volume of serum samples may be about 0.1 mlto about 2 ml, preferably about 1 ml. The volume of urine samples may beabout 10 ml to about 30 ml, preferably about 20 ml.

The term “subject” is intended to include all animals shown to orexpected to have nucleic acid-containing particles. In particularembodiments, the subject is a mammal, a human or nonhuman primate, adog, a cat, a horse, a cow, other farm animals, or a rodent (e.g. mice,rats, guinea pig, etc.). A human subject may be a normal human beingwithout observable abnormalities, e.g., a disease. A human subject maybe a human being with observable abnormalities, e.g., a disease. Theobservable abnormalities may be observed by the human being himself, orby a medical professional. The term “subject”, “patient”, and“individual” are used interchangeably herein.

The biological sample may be pre-processed before isolating nucleicacid-containing particles. In some instances, a pre-processing step ispreferred. For example, a urine sample may be pre-processed to obtainurinary nucleic acid-containing particles. The pre-processing may beachieved by techniques known in the art such as differentialcentrifugation or filtration. For example, urine samples may undergo afirst centrifugation step of about 300 g to get rid of large particlesand debris in the samples. Urine samples may then undergo a secondcentrifugation step of about 5,000 g to about 20,000 g (larger volumecentrifuged-higher k-factor) to get rid of unwanted particles that didnot pellet in the previous centrifugation step, but without pelletingnucleic acid-containing particles that are desired in the finalanalysis. After the second centrifugation step, urine samples mayfurther undergo a filtration step, e.g., 0.8 μm, 0.45 μm, or 0.22 μmfiltration step to further rid the sample of unwanted materials.Alternatively, urine samples may be pre-processed by a filtration stepwithout first undergoing the one or more of the centrifugation steps.

Generally therefore the biological sample may be pre-processed bycentrifuging at a low speed of about 100-500 g, preferably about 250-300g, to remove large unwanted particles and debris in the sample.Alternatively or additionally the biological sample may be pre-processedby centrifuging at a higher speed of about 10,000-20,000 g, preferably15,000-19,000 g, to remove unwanted particles and substances in thesample. Where both centrifugation pre-processing steps are performed,the biological sample may be centrifuged first at the lower speed andthen at the higher speed. If desired, further suitable centrifugationpre-processing steps may be carried out. For example, the step ofcentrifugation may be repeated for further pre-processing the samples.Alternatively or in addition to the one or more centrifugationpre-processing steps, the biological sample may be filtered. A filterhaving a size in the range about 0.1 to about 1.0 μm may be employed,preferably about 0.5 to about 1.0 μm, e.g. about 0.7 μm or about 0.8 μm.

The isolation step is advantageous for the extraction of high qualitynucleic acids from a biological sample for the following reasons: 1)extracting nucleic acids from particles provides the opportunity toselectively analyze disease- or tumor-specific nucleic acids, which maybe obtained by isolating disease- or tumor-specific particles apart fromother particles within the fluid sample; 2) nucleic acid-containingparticles such as microvesicles produce significantly higher yields ofnucleic acid species with higher integrity as compared to theyield/integrity obtained by extracting nucleic acids directly from thefluid sample without first isolating microvesicles; 3) scalability, e.g.to detect nucleic acids expressed at low levels, the sensitivity can beincreased by pelleting more nucleic acid-containing particles from alarger volume of serum; 4) purer nucleic acids in that protein andlipids, debris from dead cells, and other potential contaminants and PCRinhibitors are excluded from the pellets before the nucleic acidextraction step; and 5) more choices in nucleic acid extraction methodsas pellets are of much smaller volume than that of the starting serum,making it possible to extract nucleic acids from these pellets usingsmall volume column filters.

In one embodiment, the method of isolating particles from a body fluidand extracting nucleic acids from the isolated particles may comprisethe steps of: removing cells from the body fluid either by low speedcentrifugation and/or filtration though a 0.8 μm filter; centrifugingthe supernatant/filtrate at about 20,000 g for about 0.5 hour at about4° C. using about 1 ml sample volume; treating the pellet with apre-lysis solution, e.g., an RNase inhibitor and/or a pH bufferedsolution and/or a protease enzyme in sufficient quantities (as describedbelow); and lysing the pellet for nucleic acid extraction. In oneembodiment, the process of isolating particles and extracting highquality nucleic acids may be achieved within 90 minutes.

Following isolation, nucleic acid may be extracted from the pelletedparticles. To achieve this, in some embodiments, the particles may firstbe lysed. The lysis of particles such as microvesicles in the pellet andextraction of nucleic acids may be achieved with various methods knownin the art. In one embodiment, the lysis and extraction steps may beachieved using a commercially available Qiagen RNeasy Plus kit. Inanother embodiment, the lysis and extraction steps may be achieved usinga commercially available Qiagen miRNeasy kit. In yet another embodiment,the nucleic acid extraction may be achieved using phenol:chloroformaccording to standard procedures and techniques known in the art.

According to the present invention, the novel nucleic acid extractionmethods include the step of removing or mitigating adverse factors thatprevent high quality nucleic acid extraction from a biological sample.Such adverse factors are heterogeneous in that different biologicalsamples may contain various species of adverse factors. In somebiological samples, factors such as excessive DNA may affect the qualityof nucleic acid extractions from such samples. In other samples, factorssuch as excessive endogenous RNase may affect the quality of nucleicacid extractions from such samples. Many agents and methods may be usedto remove these adverse factors. These methods and agents are referredto collectively herein as an “extraction enhancement operations.”

In some instances, the extraction enhancement operation may involve theaddition of nucleic acid extraction enhancement agents to the biologicalsample. To remove adverse factors such as endogenous RNases, suchextraction enhancement agents as defined herein may include, but are notlimited to, an RNase inhibitor such as Superase-In (commerciallyavailable from Ambion Inc.) or RNaseINplus (commercially available fromPromega Corp.), or other agents that function in a similar fashion; aprotease (which may function as an RNase inhibitor); DNase; a reducingagent; a decoy substrate such as a synthetic RNA and/or carrier RNA; asoluble receptor that can bind RNase; a small interfering RNA (siRNA);an RNA binding molecule, such as an anti-RNA antibody, a basic proteinor a chaperone protein; an RNase denaturing substance, such as a highosmolarity solution, a detergent, or a combination thereof. Theseenhancement agents may exert their functions in various ways, e.g.,through inhibiting RNase activity (e.g., RNase inhibitors), through aubiquitous degradation of proteins (e.g., proteases), or through achaperone protein (e.g., a RNA-binding protein) that binds and protectsRNAs. In all instances, such extraction enhancement agents remove or atleast mitigate some or all of the adverse factors in the biologicalsample or associated with the isolated particles that would otherwiseprevent or interfere with the high quality extraction of nucleic acidsfrom the isolated particles.

For example, the extraction enhancement operation may include theaddition of an RNase inhibitor to the biological sample, and/or to theisolated particle fraction, prior to extracting nucleic acid; preferablythe RNase inhibitor has a concentration of greater than 0.027 AU (1×)for a sample equal to or more than 1 μl in volume; alternatively,greater than or equal to 0.135 AU (5×) for a sample equal to or morethan 1 μl; alternatively, greater than or equal to 0.27 AU (10×) for asample equal to or more than 1 μl; alternatively, greater than or equalto 0.675 AU (25×) for a sample equal to or more than 1 μl; andalternatively, greater than or equal to 1.35 AU (50×) for a sample equalto or more than 1 μl; wherein the 1× concentration refers to anenzymatic condition wherein 0.027 AU or more RNase inhibitor is used totreat particles isolated from 1 μl or more bodily fluid, the 5×concentration refers to an enzymatic condition wherein 0.135 AU or moreRNase inhibitor is used to treat particles isolated from 1 μl or morebodily fluid, the 10× protease concentration refers to an enzymaticcondition wherein 0.27 AU or more RNasc inhibitor is used to treatparticles isolated from 1 μl or more bodily fluid, the 25× concentrationrefers to an enzymatic condition wherein 0.675 AU or more RNaseinhibitor is used to treat particles isolated from 1 μl or more bodilyfluid, and the 50× protease concentration refers to an enzymaticcondition wherein 1.35 AU or more RNase inhibitor is used to treatparticles isolated from 1 μl or more bodily fluid. Preferably, the RNaseinhibitor is a protease, in which case, 1 AU is the protease activitythat releases folin-positive amino acids and peptides corresponding to 1μmol tyrosine per minute.

One surprising manifestation of the high quality nucleic acid extractionusing the new method of the present invention is the ability to detectin an extraction of nucleic acids from particles such as microvesiclesthe existence of significant quantities of ribosomal RNA (rRNA). Noprior studies are known to have demonstrated the detection of 18S and28S rRNAs in nucleic acid extractions from particles. On the contrary,prior studies suggested that no or little rRNA is present in nucleicacid extracts from microvcsicles (Skog et al., 2008; Taylor andGercel-Taylor, 2008; Valadi et al., 2007). See also, the productdescription of ExoMir™ kit (Bioo Scientific Corp., Austin, Tex.).

In one embodiment, the extracted nucleic acid comprises RNA. In thisinstance, the RNA is preferably reverse-transcribed into complementaryDNA (cDNA) before further amplification. Such reverse transcription maybe performed alone or in combination with an amplification step. Oneexample of a method combining reverse transcription and amplificationsteps is reverse transcription polymerase chain reaction (RT-PCR), whichmay be further modified to be quantitative, e.g., quantitative RT-PCR asdescribed in U.S. Pat. No. 5,639,606, which is incorporated herein byreference for this teaching. Another example of the method comprises twoseparate steps: a first of reverse transcription to convert RNA intocDNA and a second step of quantifying the amount of cDNA usingquantitative PCR. As demonstrated in the examples that follow, the RNAsextracted from nucleic acid-containing particles using the methodsdisclosed herein include many species of transcripts including, but notlimited to, the transcripts that correspond to those for GAPDH, BRAF,KLK3, EGFR, and ribosomal 18S rRNA.

Nucleic acid amplification methods include, without limitation,polymerase chain reaction (PCR) (U.S. Pat. No. 5,219,727) and itsvariants such as in situ polymerase chain reaction (U.S. Pat. No.5,538,871), quantitative polymerase chain reaction (U.S. Pat. No.5,219,727), nested polymerase chain reaction (U.S. Pat. No. 5,556,773),self-sustained sequence replication and its variants (Guatelli et al.,1990), transcriptional amplification system and its variants (Kwoh etal., 1989), Qb Replicase and its variants (Miele et al., 1983), cold-PCR(Li et al., 2008), BEAMing (Li et al., 2006) or any other nucleic acidamplification methods, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.Especially useful are those detection schemes designed for the detectionof nucleic acid molecules if such molecules are present in very lownumbers. The foregoing references are incorporated herein for theirteachings of these methods. In other embodiment, the step of nucleicacid amplification is not performed. Instead, the extract nucleic acidsare analyzed directly, e.g., through next-generation sequencing.

The analysis of nucleic acids present in the isolated particles isquantitative and/or qualitative. For quantitative analysis, the amounts(expression levels), either relative or absolute, of specific nucleicacids of interest within the isolated particles are measured withmethods known in the art (described below). For qualitative analysis,the species of specific nucleic acids of interest within the isolatedparticles, whether wild type or variants, are identified with methodsknown in the art.

The present invention also includes various uses of the new methods ofnucleic acid extraction from a biological sample for (i) aiding in thediagnosis of a subject, (ii) monitoring the progress or reoccurrence ofa disease or other medical condition in a subject, or (iii) aiding inthe evaluation of treatment efficacy for a subject undergoing orcontemplating treatment for a disease or other medical condition;wherein the presence or absence of one or more biomarkers in the nucleicacid extraction obtained from the method is determined, and the one ormore biomarkers are associated with the diagnosis, progress orreoccurrence, or treatment efficacy, respectively, of a disease or othermedical condition.

The one or more biomarkers can be one or a collection of geneticaberrations, which is used herein to refer to the nucleic acid amountsas well as nucleic acid variants within the nucleic acid-containingparticles. Specifically, genetic aberrations include, withoutlimitation, over-expression of a gene (e.g., an oncogene) or a panel ofgenes, under-expression of a gene (e.g., a tumor suppressor gene such asp53 or RB) or a panel of genes, alternative production of splicevariants of a gene or a panel of genes, gene copy number variants (CNV)(e.g., DNA double minutes) (Hahn, 1993), nucleic acid modifications(e.g., methylation, acetylation and phosphorylations), single nucleotidepolymorphisms (SNPs), chromosomal rearrangements (e.g., inversions,deletions and duplications), and mutations (insertions, deletions,duplications, missense, nonsense, synonymous or any other nucleotidechanges) of a gene or a panel of genes, which mutations, in many cases,ultimately affect the activity and function of the gene products, leadto alternative transcriptional splice variants and/or changes of geneexpression level, or combinations of any of the foregoing.

The determination of such genetic aberrations can be performed by avariety of techniques known to the skilled practitioner. For example,expression levels of nucleic acids, alternative splicing variants,chromosome rearrangement and gene copy numbers can be determined bymicroarray analysis (see, e.g., U.S. Pat. Nos. 6,913,879, 7,364,848,7,378,245, 6,893,837 and 6,004,755) and quantitative PCR. Particularly,copy number changes may be detected with the Illumina Infinium II wholegenome genotyping assay or Agilent Human Genome CGH Microarray (Steemerset al., 2006). Nucleic acid modifications can be assayed by methodsdescribed in, e.g., U.S. Pat. No. 7,186,512 and patent publicationWO/2003/023065. Particularly, methylation profiles may be determined byIllumina DNA Methylation OMA003 Cancer Panel. SNPs and mutations can bedetected by hybridization with allele-specific probes, enzymaticmutation detection, chemical cleavage of mismatched heteroduplex (Cottonet al., 1988), ribonuclease cleavage of mismatched bases (Myers et al.,1985), mass spectrometry (U.S. Pat. Nos. 6,994,960, 7,074,563, and7,198,893), nucleic acid sequencing, single strand conformationpolymorphism (SSCP) (Orita et al., 1989), denaturing gradient gelelectrophoresis (DGGE)(Fischer and Lerman, 1979a; Fischer and Lerman,1979b), temperature gradient gel electrophoresis (TGGE) (Fischer andLerman, 1979a; Fischer and Lerman, 1979b), restriction fragment lengthpolymorphisms (RFLP) (Kan and Dozy, 1978a; Kan and Dozy, 1978b),oligonucleotide ligation assay (OLA), allele-specific PCR (ASPCR) (U.S.Pat. No. 5,639,611), ligation chain reaction (LCR) and its variants(Abravaya ct al., 1995; Landegren et al., 1988; Nakazawa et al., 1994),flow-cytometric heteroduplex analysis (WO/2006/113590) andcombinations/modifications thereof. Notably, gone expression levels maybe determined by the serial analysis of gene expression (SAGE) technique(Velculescu et al., 1995). In general, the methods for analyzing geneticaberrations are reported in numerous publications, not limited to thosecited herein, and are available to skilled practitioners. Theappropriate method of analysis will depend upon the specific goals ofthe analysis, the condition/history of the patient, and the specificcancer(s), diseases or other medical conditions to be detected,monitored or treated. The forgoing references are incorporated hereinfor their teaching of these methods.

Many biomarkers may be associated with the presence or absence of adisease or other medical condition in a subject. Therefore, detection ofthe presence or absence of such biomarkers in a nucleic acid extractionfrom isolated particles, according to the methods disclosed herein, mayaid diagnosis of the disease or other medical condition in the subject.For example, as described in WO 2009/100029, detection of the presenceor absence of the EGFRvIII mutation in nucleic acids extracted frommicrovesicles isolated from a patient serum sample may aid in thediagnosis and/or monitoring of glioblastoma in the patient. This is sobecause the expression of the EGFRvIII mutation is specific to sometumors and defines a clinically distinct subtype of glioma (Pelloski etal., 2007). For another example, as described in WO 2009/100029,detection of the presence or absence of the TMPRSS2-ERG fusion geneand/or PCA-3 in nucleic acids extracted from microvesicles isolated froma patient urine sample may aid in the diagnosis of prostate cancer inthe patient.

Further, many biomarkers may help disease or medical status monitoringin a subject. Therefore, the detection of the presence or absence ofsuch biomarkers in a nucleic acid extraction from isolated particles,according to the methods disclosed herein, may aid in monitoring theprogress or reoccurrence of a disease or other medical condition in asubject. For example, as described in WO 2009/100029, the determinationof matrix metalloproteinase (MMP) levels in nucleic acids extracted frommicrovesicles isolated from an organ transplantation patient may help tomonitor the post-transplantation condition, as a significant increase inthe expression level of MMP-2 after kidney transplantation may indicatethe onset and/or deterioration of post-transplantation complications.Similarly, a significantly elevated level of MMP-9 after lungtransplantation, suggests the onset and/or deterioration ofbronchiolitis obliterans syndrome.

Many biomarkers have also been found to influence the effectiveness oftreatment in a particular patient. Therefore, the detection of thepresence or absence of such biomarkers in a nucleic acid extraction fromisolated particles, according to the methods disclosed herein, may aidin evaluating the efficacy of a given treatment in a given patient. Forexample, as disclosed in Table 1 in the publication by Furnari et. al.(Furnari et al., 2007), biomarkers, e.g., mutations in a variety ofgenes, affect the effectiveness of specific medicines used inchemotherapy for treating brain tumors. The identification of thesebiomarkers in nucleic acids extracted from isolated particles from abiological sample from a patient may guide the selection of treatmentfor the patient.

One aspect of the present invention is further directed to a kit for usein the new methods disclosed herein. The kit is comprised of thefollowing components: RNase inhibitor in quantity sufficient to mitigateadverse factors that prevent or might prevent high quality nucleic acidextraction; RNA purification reagent; optionally, lysis buffer;optionally, DNase; and optionally, instructions for using the foregoingreagents in the extraction of nucleic acids from isolated particles. TheRNA purification reagent helps to purify the released nucleic acids. Thelysis buffer helps to break open microvesicles so that their nucleicacid contents are released. The use of DNase may help enhance thequality of the extracted nucleic acids. The inclusion of DNase isoptional because DNase digestion may sometimes be carried out on anucleic acid purification column, as described in the second exampleunder the section “Particle isolation and nucleic acid extraction fromserum samples.” The kit may also comprise instructions that detail thesteps as appropriate for using the kit components in connection with theextraction of nucleic acids from isolated particles.

It should be understood that this invention is not limited to theparticular methodologies, protocols and reagents, described herein,which may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Examples of the disclosed subject matter are set forth below. Otherfeatures, objects, and advantages of the disclosed subject matter willbe apparent from the detailed description, figures, examples and claims.Methods and materials substantially similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentlydisclosed subject matter. Exemplary methods and materials are nowdescribed as follows.

Example 1

We obtained a 1 ml frozen serum sample from a normal, healthy humanvolunteer. The serum sample was filtered through a 0.8 μm filter(Millipore) and the filtrate was then stored at −80° C. for 24 hours.When the sample was thawed, 8 μl SuperaseIn was added. The sample wasthen centrifuged at 20,000 g (Hettich microcentrifuge) for 0.5 hour at4° C. in an angle head rotor. The supernatant was removed and discarded.The pellet was re-suspended in 1.5 ml PBS and re-centrifuged at 20,000 gfor another 0.5 hour. The supernatant was then removed and discarded.The pellet was treated with 8 μl SuperascIn (20 units/μl) for 1 minuteand then re-suspended in RLT buffer plus 10 μl/ml betamercaptoethanoland processed using the Qiagen RNeasy Plus kit which features a DNAremoval column. The nucleic acids were eluted in 16 μl nuclease-freeH₂O.

We examined the quality of the extracted nucleic acids using an RNA PicoChip on an Agilent Bioanalyzer. As shown in FIG. 1A, we detected thepresence of the 18S and 28S rRNA in the extraction. The RNA IntegrityNumber (RIN), as calculated by the Bioanalyzer's software, was 8.5. Inaddition, in the extracted nucleic acids, we detected the presence ofRNAs corresponding to the GAPDH, BRAF, and 18S RNA genes. We used 12 μlof the extracted RNA and reverse transcribed the RNA into cDNA using aSensiscript kit (Qiagen). We then used 2 μl of the resulting cDNAproduct as templates to perform Real-time PCR. The primers used for theRT-PCR are commercially available from Applied Biosystems, as follows:Human GAPDH (part number 4326317E); BRAF (part number Hs00269944_m1);18S rRNA (part number Hs99999901_s1). Each sample was run in triplicateon the PCR plate. The Ct values from the RT-PCR investigation arepresented as average±SD. The Ct values for GAPDH, BRAF and 18S rRNA are30.84±0.08, 36.76±0.22, and 15.09±0.21, respectively.

Therefore, using the new method, we were able to isolate nucleicacid-containing particles from serum samples. The nucleic acidsextracted from the pelleted particles contained 18S and 28S rRNA. Thequality of the nucleic acids produced a RIN of 8.5. Further, theextracted nucleic acids contain RNAs corresponding to at least GAPDH,BRAF and 18S rRNA genes, suggesting that the extracted nucleic acidsfrom serum particles may include RNAs corresponding to many other genes.

Example 2

We obtained a 1 ml frozen serum sample from the same normal, healthyhuman volunteer as in Example 1 and filtered the serum through a 0.8 μmfilter (Millipore) and the filtrate was then stored at −80° C. for 24hours. The frozen sample was thawed on ice, and transferred into a 1.5ml Eppendorf tube containing 8 μl SuperaseIn (Ambion Inc.). After the20,000 g, 0.5 hour centrifugation step, the supernatant was set asidefor further extraction as detailed in Example 3 below. The pellet wasused for nucleic acid extraction employing a modified miRNeasy RNAextraction protocol. This modified protocol was more efficient atcapturing the small RNAs (e.g., less than 200 nucleotides) than themanufacturer's protocol contained in the RNeasy Plus kit used in Example1.

In this modified protocol, we used a mixture of DNAse/SuperaseIn totreat the pellet (TURBO DNA-Free™ kit, Ambion). The DNase could beoptionally replaced by an on-column DNase step following the miRNcasyprotocol. This treatment removed most DNA, including DNA potentiallycoming from inside the isolated particles. These DNA may affect RNAintegrity when the extracted RNA quantity is very small. If on-columnDNase treatment is selected, the pellet is treated with 8 μL SuperaseInin 42 μL PBS. The mixture of DNase and SuperaseIn RNase inhibitor inthis particular sample was made according to the following scheme. DNase1 and DNase buffer is from TURBO DNA-Free™ kit from Ambion. SuperaseInwas at a concentration of 20 units/μL.

Per Sample:

DNase 1 2 μL DNase buffer (10X) 5 μL SuperaseIn 8 μL 1xPBS 35 μL  50 μL 

The pellet was mixed with 50 μL of the DNase/SuperaseIn mixture asmentioned above and incubated at room temperature for 20 min in thecentrifuge tube. Then 700 μl Qiazol lysis buffer (Qiagen) was added toeach sample in the centrifuge tube and mixed by pipetting up and down 15times to dissolve/re-suspend the pellet. The suspended pellet mixturewas immediately transferred to an Eppendorf tube. Further nucleic acidextraction was then performed in a PCR hood. The tube with the pelletmixture was vortexed briefly and incubated at room temperature for 2-4minutes before 140 μl chloroform was added into the tube containing themixture. The tube was then capped, shaken vigorously for 20 seconds,incubated at room temperature for 2-3 minutes, and centrifuged for 15minutes at 12,000 g at 4° C. The upper aqueous phase was transferred toa new collection tube into which, 1.5 volumes (usually 600 μl) of 100%ethanol was added and mixed thoroughly by pipetting up and down severaltimes.

Up to 700 μl of the ethanol mixture, including any precipitate that mayhave formed, was transferred into an RNeasy Micro spin column (MinElutecolumn stored @+4° C., the column comes with the RNeasy Micro kit) whichwas inserted in a 2 ml collection tube as supplied by the manufacturer,and centrifuged at 1000 g for 15 second at room temperature. Theflow-through was discarded. The centrifugation step was repeated untilall the remaining mixture had been added. Again, the flow-through wasdiscarded. The nucleic acids on the column were then washed three timesas follows: 1) 700 μL Buffer RWT was added onto the RNeasy MinElute spincolumn and centrifuged for 15 seconds at 8500 g to wash the column withthe flow-through discarded; 2) 500 μL Buffer RPE was added onto theRNeasy MinElute spin column and centrifuged for 15 seconds at 8500 g towash the column with the flow-through discarded; 3) repeat the BufferRPE wash step except that the column was centrifuged for 2 minutes at8500 g to dry the RNeasy Mini spin column membrane.

After the washing steps, the RNeasyMinElute spin column was insertedinto a new 2 ml collection tube and centrifuged at 14000 g for 5 minutesto further dry the column membrane. The dried column was inserted intoanother new 1.5 ml collection tube and 16 μL RNase-free water was addedonto the dried column membrane and incubated for 1 minute at roomtemperature. The nucleic acids were eluted by centrifugation for 1minute at 8500 g. The volume of the eluted nucleic acids was about 14μl.

We analyzed the profile of the extracted nucleic acids. As shown in FIG.1B, we detected peaks corresponding to 18S and 28S rRNAs, as well aspeaks corresponding to small RNAs with sizes between 25 and 200nucleotides. In addition, in the extracted nucleic acids, we detectedthe presence of RNAs corresponding to the GAPDH, BRAF, 18S RNA, and EGFRgenes. We used 12 μl of the extracted RNA and reverse transcribed theRNA into cDNA using VILO™ kit (Invitrogen). The reverse transcriptionreaction mixture was made according to the following scheme (Table 1).

TABLE 1 Reverse transcription reaction mixture scheme. (μl) × 1 reaction×4.4 5X VILO ™ Reaction Mix 4 17.6 10X Superscript ® Enzyme 2 8.8 MixRNA (up to 2.5 μg) 12 — Nuclease free water 2 8.8 Total volume 20

The reverse transcription was performed in a verity PCR machine underthe following conditions: 25° C. for 10 minutes, 42° C. for 70 minutes,85° C. for 5 minutes, and was held in 4° C. before the reaction wasstored at −20° C.

We then used 1 μl of the resulting cDNA product as templates to performReal-time PCR. The primers used for the RT-PCR are commerciallyavailable from Applied Biosystems, as follows: Human GAPDH (part number4326317E); BRAF (part number Hs00269944_m1); 18S rRNA (part numberHs99999901_s1); EGFR (part number HS01076088_m1). We repeated the realtime-PCR experiments two times for each gene. The Ct values are shown inTable 2.

Therefore, using the new method as disclosed in this invention, we wereable to isolate nucleic acid-containing particles from a serum sample.The nucleic acids extracted from the isolated particles contained 18Sand 28S rRNA, as well as small RNAs. Further, the extracted nucleicacids contained RNAs for at least GAPDH, BRAF and 18S rRNA genes,suggesting that the extracted nucleic acids from serum particles mayinclude RNAs corresponding to many other genes.

TABLE 2 The Ct values for the four genes GAPDH, BRAF, 18S rRNA, andEGFR. Gene Ct value GAPDH 31.12 31.07 BRAF 33.29 34.84 18S rRNA 16.4816.46 EGFR 37.05 —

Example 3

We started with the supernatant obtained in Example 2 after centrifugingthe 1 ml serum sample at 20,000 g for 0.5 hour. The supernatant wasfurther ultracentrifuged at 120,000 g for 80 minutes at 4-8° C. (OptimaMax-XP Benchtop ultracentrifuge from Beckman). The deceleration was setat 7. Nucleic acids were then extracted from the pellet following thesame protocol as detailed above in Example 2 starting from a treatmentwith DNase and SuperaseIn mixture. We analyzed the profile of theextracted nucleic acids, and performed reverse transcription and realtime PCR analysis of the same four genes as in Example 2.

As shown in FIG. 1C, more small RNAs were seen in the extracted nucleicacids. The peaks between 25 and 200 nucleotides were higher than thosein the FIG. 1B. Further the peaks shifted left in the interval between25 and 200 nucleotides, suggesting the percentage of smaller RNAs washigher than the percentage seen in the extraction from Example 2.

As in Example 2, we also detected the RNAs corresponding to the fourgenes GAPDH, BRAF, 18S rRNA, and EGFR. The Ct values are shown in Table3.

TABLE 3 The Ct values for the four genes GAPDH, BRAF, 18S rRNA, andEGFR. Gene Ct value GAPDH 34.71 34.97 BRAF — 37.18 18S rRNA 21.07 21.13EGFR 32.8 31.83

Therefore, we were able to isolate nucleic acid-containing particlesfrom the supernatant obtained in Example 2 after serum centrifugation at20,000 g for 0.5 hour. The nucleic acids extracted from the particlespelleted from the supernatant contained more abundant small RNAs thanthe nucleic acids extracted from the particles initially pelleted inExample 2. Further, the extracted nucleic acids contained RNAs for atleast GAPDH, BRAF and 18S rRNA genes, suggesting that the extractednucleic acids from supernatant may include RNAs corresponding to manyother genes.

Example 4

We started with a 24 ml serum sample from a healthy normal volunteer.The serum sample was filtered through a 0.8 m filter (Millipore) and thefiltrate was then stored at −80° C. for 24 hours. The 24 ml serum samplewas thawed and transferred into 24 tubes with 1 ml in each tube. Intoeach tube 8 μl SuperaseIn was then added and mixed with the serumsample.

We separated the 24 tubes into two groups each consisting of 12 tubes:group A and group B. For group A, the serum samples were centrifuged at20,000 g for 0.5 hour and the pellet was used for nucleic acidextraction employing a modified miRNeasy RNA extraction protocol asdescribed in Example 2. For group B, the serum samples were centrifugedat 120,000 g for 80 minutes and the pellet was used for nucleic acidextraction employing a modified miRNeasy RNA extraction protocol asdescribed in Example 2.

We analyzed the profile of the extracted nucleic acids in both group Aand group B. As shown in FIG. 3 (group A) and FIG. 4 (group B), wedetected peaks corresponding to 18S and 28S rRNAs, as well as peakscorresponding to small RNAs with sizes between 25 and 300 nucleotides inboth groups. The ratio of 28S rRNA over 18S rRNA was 0.9 and 1.2 forgroup A and group B, respectively.

We further detected the expression of many genes in the RNA extractedfrom both groups. The RNA extracted from particles pelleted from each ofthe serum samples was each reversed transcribed into cDNA using theVILO™ kit from Invitrogen as described in Example 2, and then analyzedusing the TaqMan® array 96 Human Cell Surface Markers PCR plate fromApplied Biosystems according to the manufacturer's protocol.

The Applied Biosystems assay IDs in the 96 Human Cell Surface MarkersTaqman® PCR Array are shown in Table 4.

TABLE 4 Applied Biosystems assay IDs in the 96 Human Cell SurfaceMarkers Taqman ® PCR Array ID 1 2 3 4 5 6 A Hs99999901_s1 Hs99999905_m1Hs99999909_m1 Hs99999908_m1 Hs99999903_m1 Hs99999907_m1 B Hs00233455_m1Hs00704891_s1 Hs00199894_m1 Hs00174705_m1 Hs99999192_m1 Hs00233332_m1 CHs00175568_m1 Hs00609515_m1 Hs00174796_m1 Hs01099648_m1 Hs01120071_m1Hs00174158_m1 D Hs00156390_m1 Hs00934033_m1 Hs00196191_m1 Hs00174297_m1Hs00233564_m1 Hs00269961_m1 E Hs00174762_m1 Hs00175524_m1 Hs01556595_m1Hs00164004_m1 Hs01028971_m1 Hs00153398_m1 F Hs01077044_m1 Hs00417598_m1Hs01058806 g1 Hs00219575_m1 Hs00609563_m1 Hs01106578_m1 G Hs00970273_g1Hs00233844_m1 Hs01920599_gH Hs00361185_m1 Hs02339473_g1 Hs00544819_m1 HHs00169777_m1 Hs00220767_m1 Hs00374264_g1 Hs00927900_m1 Hs00949382_m1Hs00158980_m1 ID 7 8 9 10 11 12 A Hs99999902_m1 Hs00609297_m1Hs99999910_m1 Hs99999906_m1 Hs00824723_m1 Hs99999904_m1 B Hs00233509_m1Hs00939888_m1 Hs00233515_m1 Hs01588349_m1 Hs00233533_m1 Hs02379687_s1 CHs00962186_m1 Hs00181217_m1 Hs99999100_s1 Hs00163934_m1 Hs00204397_m1Hs00198752_m1 D Hs00998119_m1 Hs00236881_m1 Hs00175478_m1 Hs00188486_m1Hs01567025_m1 Hs00233520_m1 E Hs00911250_m1 Hs03044418_m1 Hs00175210_m1Hs00923996_m1 Hs00236330_m1 Hs00758600_m1 F Hs01030384_m1 Hs00907778_m1Hs00235006_m1 Hs00158127_m1 Hs01076873_m1 Hs00174469_m1 G Hs00292551_m1Hs00159522_m1 Hs00538076_m1 Hs00941830_m1 Hs00167166_m1 Hs01573922_m1 HHs00945155_m1 Hs00533968_m1 Hs00174277_m1 Hs02576518_gH Hs01003372_m1Hs00169795_m1

The gene symbols in the 96 Human Cell Surface Markers Taqman® PCR Arrayare shown in Table 5.

TABLE 5 Gene symbols corresponding to the 96 Human Cell Surface MarkersTaqman ® PCR Array Sym- bol 1 2 3 4 5 6 7 8 9 10 11 12 A 18S GAPDH HPRT1GUSB ACTB B2M RPLP0 HMBS TBP PGK1 UBC PP1A B ALCAM C5AR1 CD160 CD163CD19 CD1A CD1C CD1D CD2 CD209 CD22 CD24 C CD244 CD247 CD28 CD37 CD38CD3D CD3G CD4 CD40 CD40LG CD5 CD6 D CD63 CD69 CD7 CD70 CD72 CD74 CD79ACD79B CD80 CD83 CD86 CD8A E CD8B CD96 CHST10 COL1A1 COL1A2 CR2 CSF1RCTLA4 DPP4 ENG FAS FCER1A F FCER2 FCGR1A; HLA- HLA- ICAM2 IL1R2B1 IL1R2IL2RA ITGA1 ITGA2 ITGA3 KLRB1 FCGR1B; A; HLA- DRA FCGR1C A29.1 G KLRC1KLRD1 KRT18 KRT5 KRT8, MS4A1 MYH10 MYH9 MYOCD NCAM1 NOS3 NT5E LOC728638H PECAM1 RETN S100A8 SELP ST6GAL1 EPCAM TEK TNFRSF4 TNFRSF8 TPSAB1;VCAM1 VWF TPSB2

As shown in Table 6, we detected expression for most of the genes on thearray. The expression levels are represented in Ct values.

TABLE 6 Gene expression levels in particles from each serum sample. CTCT Well Target Gene Name (Group A) (Group B) A1 18S-Hs99999901_s1 12.1412.06 A2 GAPDH-Hs99999905_m1 26.47 26.18 A3 HPRT1-Hs99999909_m1 31.1830.95 A4 GUSB-Hs99999908_m1 33.21 32.77 A5 ACTB-Hs99999903_m1 25.0925.14 A6 B2M-Hs99999907_m1 22.32 22.30 A7 RPLP0-Hs99999902_m1 25.3824.97 A8 HMBS-Hs00609297_m1 32.67 32.42 A9 TBP-Hs99999910_m1 33.51 32.85A10 PGK1-Hs99999906_m1 28.39 28.38 A11 UBC-Hs00824723_m1 27.28 27.30 A12PPIA-Hs99999904_m1 26.57 26.47 B1 ALCAM-Hs00233455_m1 35.85 34.58 B2C5AR1-Hs00704891_s1 28.27 28.29 B3 CD160-Hs00199894_m1 34.93 34.82 B4CD163-Hs00174705_m1 34.85 35.00 B5 CD19-Hs99999192_m1 33.76 33.24 B6CD1A-Hs00233332_m1 Undeter- Undeter- mined mined B7 CD1C-Hs00233509_m135.13 34.70 B8 CD1D-Hs00939888_m1 33.79 34.10 B9 CD2-Hs00233515_m1 30.9130.90 B10 CD209-Hs01588349_m1 37.52 Undeter- mined B11CD22-Hs00233533_m1 29.82 29.99 B12 CD24-Hs02379687_s1 30.55 30.25 C1CD244-Hs00175568_m1 30.85 31.50 C2 CD247-Hs00609515_m1 30.64 30.44 C3CD28-Hs00174796_m1 33.23 32.94 C4 CD37-Hs01099648_m1 30.59 30.41 C5CD38-Hs01120071_m1 34.69 34.30 C6 CD3D-Hs00174158_m1 30.58 30.36 C7CD3G-Hs00962186_m1 31.54 31.48 C8 CD4-Hs00181217_m1 33.24 33.65 C9CD40-Hs99999100_s1 33.08 33.12 C10 CD40LG-Hs00163934_m1 33.71 33.64 C11CD5-Hs00204397_m1 33.41 33.91 C12 CD6-Hs00198752_m1 33.47 33.99 D1CD63-Hs00156390_m1 29.62 29.15 D2 CD69-Hs00934033_m1 32.52 32.32 D3CD7-Hs00196191_m1 31.64 31.83 D4 CD70-Hs00174297_m1 36.99 38.20 D5CD72-Hs00233564_m1 32.82 32.68 D6 CD74-Hs00269961_m1 28.93 28.71 D7CD79A-Hs00998119_m1 31.48 31.20 D8 CD79B-Hs00236881_m1 31.93 32.11 D9CD80-Hs00175478_m1 37.80 Undeter- mined D10 CD83-Hs00188486_m1 33.1131.97 D11 CD86-Hs01567025_m1 35.40 35.20 D12 CD8A-Hs00233520_m1 31.3831.89 E1 CD8B-Hs00174762_m1 33.93 33.52 E2 CD96-Hs00175524_m1 34.1433.82 E3 CHST10-Hs01556595_m1 37.54 Undeter- mined E4COL1A1-Hs00164004_m1 Undeter- Undeter- mined mined E5COL1A2-Hs01028971_m1 36.61 Undeter- mined E6 CR2-Hs00153398_m1 35.3134.87 E7 CSF1R-Hs00911250_m1 37.17 36.63 E8 CTLA4-Hs03044418_m1 36.2437.17 E9 DPP4-Hs00175210_m1 33.75 34.37 E10 ENG-Hs00923996_m1 32.3431.41 E11 FAS-Hs00236330_m1 33.77 33.55 E12 FCER1A-Hs00758600_m1 37.2637.73 F1 FCER2-Hs01077044_m1 34.15 33.91 F2 FCGR1A; FCGR1B; FCGR1C-33.29 32.87 Hs00417598_m1 F3 HLA-A; HLA-A29.1- 27.51 27.40 Hs01058806_g1F4 HLA-DRA-Hs00219575_m1 27.75 27.69 F5 ICAM2-Hs00609563_m1 30.65 30.61F6 IL12RB1-Hs01106578_m1 32.77 32.87 F7 IL1R2-Hs01030384_m1 32.45 31.97F8 IL2RA-Hs00907778_m1 35.00 34.78 F9 ITGA1-Hs00235006_m1 31.78 32.76F10 ITGA2-Hs00158127_m1 37.80 Undeter- mined F11 ITGA3-Hs01076873_m137.20 37.14 F12 KLRB1-Hs00174469_m1 28.43 28.37 G1 KLRC1-Hs00970273_g134.15 35.50 G2 KLRD1-Hs00233844_m1 31.96 32.27 G3 KRT18-Hs01920599_gH32.59 31.41 G4 KRT5-Hs00361185_m1 37.99 Undeter- mined G5 KRT8;LOC728638- Undeter- 38.60 Hs02339473_g1 mined G6 MS4A1-Hs00544819_m130.92 31.20 G7 MYH10-Hs00292551_m1 35.32 34.88 G8 MYH9-Hs00159522_m126.30 26.40 G9 MYOCD-Hs00538076_m1 Undeter- Undeter- mined mined G10NCAM1-Hs00941830_m1 36.99 35.55 G11 NOS3-Hs00167166_m1 37.07 36.85 G12NT5E-Hs01573922_m1 33.57 33.26 H1 PECAM1-Hs00169777_m1 30.28 30.28 H2RETN-Hs00220767_m1 35.14 35.08 H3 S100A8-Hs00374264_g1 25.88 25.40 H4SELP-Hs00927900_m1 30.85 30.65 H5 ST6GAL1-Hs00949382_m1 31.55 31.57 H6EPCAM-Hs00158980_m1 37.58 36.23 H7 TEK-Hs00945155_m1 30.68 32.30 H8TNFRSF4-Hs00533968_m1 33.96 35.10 H9 TNFRSF8-Hs00174277_m1 35.19 34.99H10 TPSAB1; TPSB2- Undeter- Undeter- Hs02576518_gH mined mined H11VCAM1-Hs01003372_m1 36.27 34.87 H12 VWF-Hs00169795_m1 36.39 35.45

We compared the Ct values between the two groups for each of the genestested and found that the mRNA content in the two groups was verysimilar. Therefore, we were able to isolate nucleic acid-containingparticles from the serum sample by centrifugation at either 20,000 g for0.5 hour or 120,000 g for 80 minutes. The nucleic acids extracted fromthe isolated particles contained both 18S and 28S rRNA. In addition, themRNA content obtained with a 20,000 g centrifugation speed was similarto the mRNA content obtained with a 120,000 g centrifugation speed.Further, the extracted nucleic acids from each of the pellets containedmRNAs corresponding to most of the genes tested using the Taqman array.

Example 5 Particle Isolation and Nucleic Acid Extraction from UrineSamples

We started with a 10 ml spot urine sample from normal, healthy humanvolunteers. The sample had been stored at 4° C. for a week. The urinesample was filtered through 0.8 μm filters (Nalgene). The filtrate wasthen centrifuged at 20,000 g for 1 hour at 4° C. in an angle head rotor.The supernatant was removed and discarded. The pellets were lysed in RLTbuffer plus 10 μl/ml betamercaptoethanol and processed using the QiagenRNeasy Plus kit. The nucleic acids were eluted in 16 μl nuclease-freeH₂O.

We examined the quality of the extracted nucleic acids using an AgilentBioanalyzer. As shown in FIG. 2, we detected the presence of the 18S and28S rRNA in the extractions. The RNA Integrity Number (RIN), ascalculated by the Bioanalyzer's software, was 9.1. In addition, in theextracted nucleic acids, we detected the presence of RNAs correspondingto the GAPDH, KLK3, and 18S RNA genes. We reverse transcribed 12 μl ofthe extracted RNA into cDNA using a Sensiscript kit (Qiagen). We thenused 2 μl of the resulting cDNA product as templates to performReal-time PCR. The primers used for the RT-PCR are commerciallyavailable from Applied Biosystems, as follows: Human GAPDH (part number4326317E); KLK3 (part number Hs03063374_m1); 18S rRNA (part numberHs99999901_s1). Each sample was run in triplicate on the PCR plate. TheCt values from the RT-PCR investigation are presented as average±SD. TheCt values for GAPDH, KLK3 and 18S rRNA are 26.96±0.02, 30.18±0.01, and12.22±0.15, respectively.

Therefore, using the new method as disclosed in this invention, we wereable to isolate nucleic acid-containing particles from urine samples.The nucleic acids extracted from the pelleted particles contained 18Sand 28S rRNA. The quality of the nucleic acids produced a RIN of 9.1.Further, the extracted nucleic acids contain RNAs for at least GAPDH,KLK3 and 18S rRNA genes, suggesting that the extracted nucleic acidsfrom urine particles may include RNAs corresponding to many other genes.

While the present invention has been disclosed with reference to certainembodiments, numerous modifications, alterations, and changes to thedescribed embodiments are possible without departing from the full scopeof the invention, as described in the appended specification and claims.

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1. A method for extracting nucleic acids from a biological sample,comprising the steps of: a. isolating nucleic acid-containing particlesfrom the biological sample by one or more centrifugation procedures,wherein none of the centrifugation procedures are performed at a speedexceeding about 200,000 g; b. performing one or more steps to mitigateadverse factors that prevent or might prevent high quality nucleic acidextraction; and c. extracting nucleic acids from the isolated particles.2. The method of claim 1, wherein the nucleic acid-containing particlesare microvesicles, RNA-protein complexes, DNA-protein complexes, or acombination of any of microvesicles, RNA-protein complexes, andDNA-protein complexes.
 3. The method of claim 1, wherein the nucleicacid-containing particles are microvesicles.
 4. The method of claim 1,wherein the nucleic acid-containing particles are RNA-protein complexes.5. The method of claim 1, wherein the nucleic acid-containing particlesare DNA-protein complexes.
 6. The method of claim 1, wherein all of thecentrifugation procedures are performed at speeds of about 2,000 g toabout 200,000 g.
 7. The method of claim 1, wherein none of thecentrifugation procedures are performed at a speed exceeding about50,000 g.
 8. The method of claim 1, wherein none of the centrifugationprocedures are performed at a speed exceeding about 20,000 g.
 9. Themethod of claim 1, wherein the biological sample is a body fluid. 10.The method of claim 9, wherein the body fluid is a serum, urine, orspinal fluid sample from a subject.
 11. The method of claim 10, whereinthe subject is a human or other mammal.
 12. The method of claim 1,wherein the extracted nucleic acids comprise RNA, DNA, or both RNA andDNA.
 13. The method of claim 1, wherein the extracted nucleic acidscomprise one or more nucleic acids having a sequence more than 90%homologous to the nucleic acid sequence corresponding to any of thegenes consisting of EGFR, BRAF, KLK3, 18S, GAPDH, HPRT1, GUSB, ACTB,B2M, RPLP0, HMBS, TBP, PGK1, UBC, PPIA, ALCAM, C5AR1, CD160, CD163,CD19, CD1A, CD1C, CD1D, CD2, CD209, CD22, CD24, CD244, CD247, CD28,CD37, CD38, CD3D, CD3G, CD4, CD40, CD4OLG, CDS, CD6, CD63, CD69, CD7,CD70, CD72, CD74, CD79A, CD79B, CD80, CD83, CD86, CD8A, CD8B, CD96,CHST10, COL1A1, COL1A2, CR2, CSF1R, CTLA4, DPP4, ENG, FAS, FCER1A,FCER2, FCGR1A/FCGR1B/FCGR1C, HLA-A/HLA-A29.1, HLA-DRA, ICAM2, IL 12RB1,IL1R2, IL2RA, ITGA1, ITGA2, ITGA3, KLRB1, KLRC1, KLRD1, KRT18, KRT5,KRT8/LOC728638, MS4A1, MYH10, MYH9, MYOCD, NCAM1, NOS3, NT5E, PECAM1,RETN, S100A8, SELP, ST6GAL1, EPCAM, TEK, TNFRSF4, TNFRSF8, TPSAB1/TPSB2,VCAM1, or VWF.
 14. The method of claim 1, wherein 18S and 28S rRNAs aredetectable in the extracted nucleic acids.
 15. The method of claim 13,wherein the ratio of the amount of 18S rRNA to the amount of 28S rRNA,as detected in the extracted nucleic acids, is about 0.5 to about 1.0.16. The method of claim 13, wherein the ratio of the amount of 18S rRNAto the amount of 28S rRNA, as detected in the extracted nucleic acids,is about 0.5.
 17. The method of claim 1, wherein step (b) is achieved bytreating the biological sample and/or the isolated particles with DNase,RNase inhibitor, or DNase and RNase inhibitor.
 18. The method of claim1, wherein step (b) comprises a step of treating the biological samplewith RNase inhibitor before isolating the particles.
 19. A nucleic acidsample obtained by the method of claim
 1. 20. The use of the method ofclaim 1 for aiding in the diagnosis of a subject, wherein the presenceor absence of a biomarker within the extracted nucleic acids isdetermined, and said biomarker is associated with a disease or othermedical condition in the subject.
 21. The use of the method of claim 1for monitoring the progress or reoccurrence of a disease or othermedical condition in a subject, wherein the presence or absence of abiomarker within the extracted nucleic acids is determined, and saidbiomarker is associated with the progress or reoccurrence of a diseaseor other medical condition in the subject.
 22. The use of the method ofclaim 1 for aiding in the evaluation of treatment efficacy for a subjectundergoing or contemplating treatment for a disease or other medicalcondition, wherein the presence or absence of a biomarker within theextracted nucleic acids is determined, and said biomarker is associatedwith treatment efficacy for the subject undergoing or contemplatingtreatment for a disease or other medical condition.
 23. The use of claim20, wherein the biomarker within the extracted nucleic acid is a nucleicacid corresponding to any one or more of the genes consisting of EGFR,BRAF, KLK3, 18S, GAPDH, HPRT1, GUSB, ACTB, B2M, RPLP0, HMBS, TBP, PGK1,UBC, PPIA, ALCAM, CSAR1, CD160, CD163, CD19, CD1A, CD1C, CD1D, CD2,CD209, CD22, CD24, CD244, CD247, CD28, CD37, CD38, CD3D, CD3G, CD4,CD40, CD4OLG, CDS, CD6, CD63, CD69, CD7, CD70, CD72, CD74, CD79A, CD79B,CD80, CD83, CD86, CD8A, CD8B, CD96, CHST10, COL1A1, COL1A2, CR2, CSF1R,CTLA4, DPP4, ENG, FAS, FCER1A, FCER2, FCGR1A/FCGR1B/FCGR1C,HLA-A/HLA-A29.1, HLA-DRA, ICAM2, IL 12RB1, IL1R2, IL2RA, ITGA1, ITGA2,ITGA3, KLRB1, KLRC1, KLRD1, KRT18, KRT5, KRT8/LOC728638, MS4A1, MYH10,MYH9, MYOCD, NCAM1, NOS3, NT5E, PECAM1, RETN, S100A8, SELP, ST6GAL1,EPCAM, TEK, TNFRSF4, TNFRSF8, TPSAB1/TPSB2, VCAM1, or VWF.
 24. A kit foruse in the method of claim 1, comprising the following components: (a)RNase inhibitor in a quantity sufficient to mitigate adverse factorsthat prevent or might prevent high quality nucleic acid extraction; (b)RNA purification reagent; (c) optionally, lysis buffer; (d) optionally,DNase; and (e) optionally, instructions for using the foregoing reagentsin the extraction of nucleic acids from isolated particles.